With the availability of a burgeoning sequence databases, genomic applications demand faster and more efficient methods for the global screening of protein expression in cells. However, the complexity of the cellular proteome expands substantially if protein post-translational modifications are also taken into account.
Dynamic post-translational modification of proteins is important for maintaining and regulating protein structure and function. Among the several hundred different types of post-translational modifications characterized to date, protein phosphorylation plays a prominent role. Enzyme-catalyzed phosphorylation and de-phosphorylation of proteins is a key regulatory event in the living cell. Complex biological processes such as cell cycle, cell growth, cell differentiation and cell metabolism are orchestrated and tightly controlled by reversible phosphorylation events which modulate protein activity, stability, interaction and localization. Perturbations in phosphorylation states of proteins, e.g. by mutations which generate constitutively active or inactive protein kinases and phosphatases, play a prominent role in oncogenesis. Comprehensive analysis and identification of phosphoproteins, combined with exact localization of phosphorylation sites in those proteins (‘phosphoproteomics’) is a prerequisite for understanding complex biological systems and the molecular features leading to disease.
It is estimated that ⅓ of all proteins present in a mammalian cell are phosphorylated and that kinases, enzymes responsible for that phosphorylation, constitute about 1–3% of the expressed genome. Organisms use reversible phosphorylation of proteins to control many cellular processes including signal transduction, gene expression, the cell cycle, cytoskeletal regulation and apoptosis. A phosphate group can modify serine, threonine, tyrosine, histidine, arginine, lysine, cysteine, glutamic acid and aspartic acid residues. However, the phosphorylation of hydroxyl groups at serine (90%), threonine (10%), or tyrosine (0.05%) residues are the most prevalent, and are involved, along with other processes, in metabolism, cell division, cell growth, and cell differentiation. Because of the central role of phosphorylation in the regulation of life, much effort has been focused on the development of methods for characterizing protein phosphorylation.
The identification of phosphorylation sites on a protein is complicated by the facts that proteins are often only partially phosphorylated and that they are often present only at very low levels. Therefore techniques for identifying phosphorylation sites should preferably work in the low picomole to sub-picomole range.
Traditional methods for analyzing O-phosphorylation sites involve incorporation of 32P into cellular proteins via treatment with radiolabeled ATP. The radioactive proteins can be detected during subsequent fractionation procedures (e.g. two-dimensional gel electrophoresis or high-performance liquid chromatography [HPLC]). Proteins thus identified can be subjected to complete hydrolysis and the phospho-amino acid content determined. The site(s) of phosphorylation can be determined by proteolytic digestion of the radiolabeled protein, separation and detection of phosphorylated peptides (e.g. by two-dimensional peptide mapping), followed by peptide sequencing by Edman degradation. These techniques can be tedious, require significant quantities of the phosphorylated protein and involve the use of considerable amounts of radioactivity.
In recent years, mass spectrometry (MS) has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The most widely used method for selectively enriching phosphopeptides from mixtures is immobilized metal affinity chromatography (IMAC). In this technique, metal ions, usually Fe3+ or Ga3+, are bound to a chelating support. Phosphopeptides are selectively bound because of the affinity of the metal ions for the phosphate moiety. The phosphopeptides can be released using high pH or phosphate buffer, the latter usually requiring a further desalting step before MS analysis. Limitations of this approach include possible loss of phosphopeptides due to their inability to bind to the IMAC column, difficulty in the elution of some multiply-phosphorylated peptides, and background from unphosphorylated peptides (typically acidic in nature) which also have some affinity for immobilized metal ions. Two types of chelating resin are commercially available, one using iminodiacetic acid and the other using nitrilotriacetic acid. Some groups have observed that iminodiacetic acid resin is less specific than nitrilotriacetic acid, whereas another study reported little difference between the two. Several studies have examined off-line MS analysis of IMAC-separated peptides.
Recently, two groups have described protocols to achieve this goal. Oda et al. (Nat Biotechnol. 2001 19:379–82) start with a protein mixture in which cysteine reactivity is removed by oxidation with performic acid. Base hydrolysis is used to induce -elimination of phosphate from phosphoserine and phosphothreonine, followed by addition of ethanedithiol to the alkene. The resulting free sulflhydryls are coupled to biotin, allowing purification of phosphoproteins by avidin affinity chromatography. Following elution of phosphoproteins and proteolysis, enrichment of phosphopeptides is carried out by a second round of avidin purification. Disadvantages of this approach include the failure to detect phosphotyrosine containing peptides and the generation of diastereoisomers in the derivatization step.
The approach suggested by Zhou et al. (Nat Biotechnol 2001 19:375–378) circumvents these problems but involves a six step derivatization/purification protocol for tryptic peptides which requires more than 13 hrs to complete and affords only a 20% yield from picomoles of phosphopeptide starting material. The method begins with a proteolytic digest which has been reduced and alkylated to eliminate reactivity from cysteine residues. Following N-terminal and C-terminal protection, phosphoramidate adducts at phosphorylated residues are formed by carbodiimide condensation with cystamine. The free sulfhydryl groups produced from this step are covalently captured onto glass beads coupled to iodoacetic acid. Elution with trifluoroacetic acid then regenerates phosphopeptides for analysis by mass spectrometry.